The cubitus interruptus (ci) locus of Drosophila melanogaster is located proximally on chromosome 4. In ci mutants cubital wing veins are interrupted or absent. We have cloned this locus using a gypsy element associated with the ci1 mutation. Analysis of all extant ci mutations reveals that they contain conspicuous molecular alterations within a 13.7 kb region. Of the four homozygous viable mutations, three (ci1, ci361, ciw) have single insertions, while one (ci57g) has a small deletion, all located within a more restricted 1 kb region. The dominant mutations, ciD and Ce2 each contain two insertions within the 13.7 kb region. All these molecular alterations are located upstream of a transcript previously associated with the ciD mutation and thought to derive from a segment polarity gene. We induced revertants of the dominant ci phenotype (wing vein interruption) in ciD and found molecular alterations in this transcript (the ci+ transcript) in two revertant alleles, thereby demonstrating this transcript's involvement in the ci phenotype. The locations of the molecular alterations, together with the results of the ciD reversion experiment, provide a connection between the dominant and recessive ci mutations and argue that all are likely to be alleles of the same complex locus, ci, not two separate loci as previously proposed. The ci phenotype of dominant and recessive mutations can be explained by inappropriate expression of the ci+ transcript in the posterior wing compartment where the cubital vein is affected, while loss of ci+ function generates recessive lethality. Lack of repression of ci+ transcription, through a pairing-dependent, trans-acting silencer element, can explain the unusual position effects associated with ci (the Dubinin effect).